Methods and compositions for modulating AAV infection
a technology of aav infection and composition, applied in the field of methods and compositions for modulating aav infection, can solve the problems of poorly understood basic aspects of aav cellular entry, and achieve the effects of reducing increasing the level, and enhancing the permissiveness of the target cell
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example 1
[0341]An unbiased, haploid genetic screen was performed to identify critical players in AAV serotype 2 (AAV2) infection. The most significantly enriched gene of the screen encodes a type-I transmembrane protein, KIAA0319L (hereafter referred to as “adeno-associated virus receptor” or “AAVR”). The experiments disclosed herein show that AAVR is a protein capable of rapidly endocytosing from the plasma membrane and trafficking to the trans-Golgi network. The experiments disclosed herein further show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently blocked AAV2 infection, that genetic ablation of AAVR rendered a wide range of mammalian cell types highly resistant to AAV2 infection, and that AAVR serves as a critical host factor for all AAV serotypes tested, including AAV1, 3B, 5, 6, 8 and 9. The importance of AAVR for in vivo gene delivery is demonstrated by the robust resistance of AAVR− / − mice to AAV infection. Collectively, the data presented here...
example 2
[0372]FIG. 14 shows that delivery of AAVR protein (e.g., using AAVR-containing gesicles) can restore AAV infection in HeLa AAVR-KO cells (which are cells knocked out for AAVR, and therefore do not express functional endogenous AAVR protein). “Uninfected” and “WT 293” are negative and positive controls, respectively. “None” is the level of infection observed in AAVR-KO cells (confirming that AAVR is an important receptor for AAV infection). “+gAAVR” is the level of infection observed when AAVR-KO cells are treated with gesicles (VSV-G induced microvesicles) that include AAVR (and AAVR is thereby delivered to the cells). Delivery of exogenous AAVR protein can therefore vastly increase a cell's permissiveness to AAV infection. “+gGFP” is a negative control in which GFP protein (instead of AAVR) is delivered to cells via gesicles. The top bar graph shows % of AAV2 infection on the Y axis while the bottom bar graph MFI (Mean Fluorescence Intensity) measured upon infection with AAV2.
[0373...
example 3
[0374]To validate AAVR's role in AAV2 infection, CRISPR / Cas9 genome engineering was used to generate isogenic AAVR knock-out cell lines (AAVRKO) in a panel of cell types representing various human and murine tissues. FIG. 16 provides data showing that isogenic AAVR knock-out cell lines (AAVRKO) in a panel of cell types representing various human and murine tissues exhibited low permissiveness to infection by AAV2. Moreover, gesicles containing AAVR delivered to the AAVR-KO cells facilitated AAV2 infection. In contrast, as a negative control, gesicles containing GFP did not facilitate AAV2 infection. Thus, AVVR plays a critical role in AAV infection in a variety of different mammalian cell types.
[0375]FIG. 17 provides data showing that overexpression of AAVR enhanced AAV2 and AAV6 infection in K562 cells. Thus, the addition of AAVR to cells increases their permissiveness to AAV infection, and the increased permissiveness is not limited to any particular AAV serotype.
[0376]FIG. 18. Pr...
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